+Open data
-Basic information
Entry | Database: PDB / ID: 8h4u | ||||||
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Title | Cryo-EM structure of a riboendonuclease | ||||||
Components | CRISPR-associated endonuclease Cas9 | ||||||
Keywords | RNA / riboendonuclease | ||||||
Function / homology | Uncharacterized protein Function and homology information | ||||||
Biological species | Thermoclostridium caenicola (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||
Authors | Li, Z. / Wang, F. | ||||||
Funding support | 1items
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Citation | Journal: J Mol Biol / Year: 2023 Title: Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola. Authors: Feng Wang / Chendi Zhang / Haijiang Xu / Wanting Zeng / Lixin Ma / Zhuang Li / Abstract: The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from ...The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8h4u.cif.gz | 189.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8h4u.ent.gz | 145.5 KB | Display | PDB format |
PDBx/mmJSON format | 8h4u.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8h4u_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 8h4u_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 8h4u_validation.xml.gz | 38.3 KB | Display | |
Data in CIF | 8h4u_validation.cif.gz | 57.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/h4/8h4u ftp://data.pdbj.org/pub/pdb/validation_reports/h4/8h4u | HTTPS FTP |
-Related structure data
Related structure data | 34484MC 8ewgC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 143582.641 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermoclostridium caenicola (bacteria) / Gene: SAMN05444373_102315 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1M6GDI0 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Apo-form riboendonuclease / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Thermoclostridium caenicola (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm |
Image recording | Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134153 / Symmetry type: POINT | ||||||||||||||||||||||||
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